Role of airway epithelial cells in host defense against pulmonary fungal pathogens

 描述(申请人提供)：呼吸道上皮细胞位于环境和底层肺组织之间的交界处。肺上皮形成抵抗环境侮辱的物理屏障，并参与防御入侵的细菌、病毒和真菌。尽管上皮细胞在抗菌和抗病毒免疫中的作用已被证实，但肺上皮在真菌免疫中的作用却知之甚少。这一建议通过研究呼吸道上皮如何协调宿主对医学上重要真菌的防御来填补这一知识空白。为了验证呼吸道上皮细胞支持产生对真菌的适应性免疫的假设，提出了3个目标。第一, 我们将评估在小鼠感染皮炎芽孢杆菌、组织胞浆菌或烟曲霉菌期间选择性地消融呼吸道上皮细胞功能的肺部真菌清除情况(目标1)。其次，通过使用AIM 1的真菌感染模型之一，我们将识别识别真菌并触发炎症介质产生的上皮细胞受体(AIM 2)。Toll样受体(Toll-like，TLR)和C型凝集素(C型lectin，CLR)受体介导真菌识别，这些受体家族的成员表达在上皮细胞上。为了分析真菌识别的机制，我们将TLR和CLR缺陷小鼠的原代呼吸道上皮细胞与皮炎假单胞菌、荚膜假单胞菌或烟熏假单胞菌共同培养，并测定培养上清中的趋化因子。这种方法将揭示趋化因子生产中的缺陷，这些缺陷源于TLR和/或CLR缺陷。第三，我们将确定上皮细胞如何影响下游免疫反应的发展，例如在抗真菌免疫中至关重要的CD4+T辅助(Th)细胞的启动(目标3)。为了解决这个问题，我们将分析TCR转基因真菌特异性Th细胞在感染小鼠中的表型和功能，这些小鼠是通过基因工程表现出呼吸道上皮细胞功能受损的。上皮细胞可能通过控制启动Th细胞应答的树突状细胞(DC)的特性间接影响Th细胞应答。因此，我们将分析上皮细胞受损小鼠体外感染时肺DC的数量、表型和功能，以确定上皮细胞衍生的趋化因子如何影响DC的募集和激活。这项工作将利用包括转基因小鼠、细胞分类、微流体和活细胞成像在内的尖端工具，并将在一个领先的实验室和研究肺部对真菌的免疫的中心进行。除了调查重要的研究问题外，这项工作还将为申请者提供 最先进的研究培训和教学技巧。完成培训后，我们将了解呼吸道上皮细胞如何感知真菌病原体(S)，由此产生的可溶性信号如何影响DC，从而塑造CD+T细胞反应的发展，以及这些事件的总和如何促进感染的解决。了解呼吸道上皮细胞如何动员抗真菌免疫将对免疫受损患者的抗真菌疫苗和基于免疫的治疗的开发具有意义，在免疫受损的患者中，真菌感染很常见，而且往往是致命的。

 DESCRIPTION (provided by applicant): Airway epithelial cells are located at the interface between the environment and underlying lung tissue. The lung epithelium forms a physical barrier against environmental insults and participates in defense against invading bacteria, viruses and fungi. Although the contribution of epithelial cells to antibacterial and antiviral immunity is well established, the role of lung epithelium in immunity to fungi is poorly understood. This proposal fills this knowledge gap by studying how the airway epithelium orchestrates host defense against medically important fungi. To test the hypothesis that airway epithelial cells underpin the generation of adaptive immunity to fungi, 3 aims are proposed. First, we will assess fungal clearance from the lung where airway epithelial cell function is selectively ablated (Aim 1) during murine infection with Blastomyces dermatitidis, Histoplasma capsulatum, or Aspergillus fumigatus. Second, by using one of the fungal infection models from Aim 1, we will identify the epithelial cell receptors that recognize fungi and trigger production of inflammatory mediators (Aim 2). Toll-like (TLR) and C-type lectin (CLR) receptors mediate fungal recognition and members of these receptor families are expressed on epithelial cells. To dissect the mechanisms of fungal recognition, we will co-culture primary airway epithelial cells from TLR- and CLR-deficient mice with B. dermatitidis, H. capsulatum or A. fumigatus and measure chemokines in the supernatant. This approach will unveil defects in chemokine production that stem from TLR and/or CLR deficiencies. Third, we will define how epithelial cells impact the development of downstream immune responses, such as the priming of CD4+ T helper (Th) cells (Aim 3) that is paramount in antifungal immunity. To address this question, we will analyze the phenotype and function of TCR transgenic, fungus-specific Th cells in infected mice genetically engineered to exhibit impaired airway epithelial cell function. Epithelial cells may influence Th cell responses indirectly by controlling the properties of dendritic cells (DC) that initiate Th cell responses. Thus, we will analyze the numbers, phenotype and function of lung DC ex-vivo upon infection of epithelial cell-impaired mice to ascertain how epithelial cell-derived chemokines affect the recruitment and activation of DC. This work will take advantage of cutting-edge tools including transgenic mice, cell sorting, microfluidics and live cell imaging and will be performed in a leading laboratory and center for the study of lung immunity to fungi. In addition to investigating vital research questions, the work will provide the applicant state-of the-art research training and didactic teaching skills. Upon completion of the training program, we will know how airway epithelial cells sense fungal pathogen(s), how the resulting soluble signals influence DC that shape the development of CD4+ T cell responses, and how the sum of these events promote the resolution of infection. Understanding how respiratory epithelial cells mobilize antifungal immunity will have implications for the development of antifungal vaccines and immune-based therapies in immune-compromised patients, where fungal infections are common and often fatal.